RNA extraction
One ml RNAiso plus reagent (Takara, Dalian, China) was added to cells on
100mm Petri dish. Cell lysates were collected and incubated at room
temperature for 5min. Cells were then centrifuged at 13500 ×g for 5min
at 4°C. A 200μL of CHCl3 was added, followed by 30sec
mixing and 5min incubation at RT, samples were centrifuged at 13500 ×g
for 15min at 4°C to separate RNA into aqueous phase. Aqueous phase
(about 600ul) was transferred to a new tube and RNA was precipitated
with 750 μL of absolute isopropanol at RT for 10min and then centrifuged
at 13500 ×g for 10min at 4°C. Precipitate was washed with 1mL 70%
ethanol, followed by centrifugation at 13500 ×g for 5min at 4°C. RNA
pellet was resuspended in 50μL of DEPC-treated water. RNA concentrations
were determined using NanoDrop 2000 (Thermo Fisher Scientific, USA). RNA
integrity was checked on 0.8% agarose gel.